JP4793265B2 - Compound WS727713 - Google Patents

Abstract

The present invention relates to a new compound useful as a modulator of melanocortin receptors. In particular, the present invention relates to a compound WS727713, a process for production of the compound by culturing, in a culture medium, a WS727713-producing strain belonging to Pseudonocardia and recovering the compound from a culture broth, a pharmaceutical composition containing the compound, and uses of the compound.

Description

  The present invention relates to novel compounds useful as drugs or cosmetics, methods for producing them, and pharmaceutical compositions containing them.

  Melanocortin (MC) is a group of peptide hormones derived from post-translational modifications by enzymatic cleavage of preprohormones. MCs include adrenocorticotropic hormone (ACTH) and melanocyte stimulating hormone (MSH) such as α-MSH, β-MSH and γ-MSH. These MCs regulate various physiological functions through membrane MC receptors (MC-R). Five MC-Rs (MC1-R, MC2-R, MC3-R, MC4-R and MC5-R) have been cloned and characterized. MC1-R, first discovered as an α-MSH receptor, is expressed in melanocytes of the integument and is involved in epidermal melanin pigmentation and animal color.

  Recently, it was discovered that MC1-R is involved in pain and inflammation, and MC1-R mRNA is expressed in inflammatory cells such as neutrophils or monocytes. MC1-R seems to explain the inhibitory action of α-MSH on nitric oxide production and neutrophil migration in monocytes. Evidence has been shown that α-MSH molecules reduce inflammatory responses in animal models of inflammatory responses in humans. α-MSH reduced general stimulus-induced inflammation in mouse skin. Α-MSH suppressed carrageenan-induced mouse paw edema. Thus, MC1-R agonists are expected to be useful as drugs for inflammatory responses.

The present invention relates to novel compounds useful as modulators of melanocortin receptors, methods for their production, and pharmaceutical or cosmetic compositions containing them.
More particularly, the present invention relates to melanocortin receptor agonist compounds having a reducing effect on inflammatory responses.

  The inventors of the present invention have also found that melanocortin receptor modulators such as WS727713 have a potent anti-inflammatory effect. Therefore, melanocortin receptor modulators such as WS727713 are useful as active ingredients in anti-inflammatory agents, and include ischemia / reperfusion injury, brain inflammatory disease, renal inflammatory disease, hepatitis, septic / septic Shock, hypoxic shock, acute respiratory distress syndrome (ARDS), rheumatoid arthritis (RA), gouty arthritis, aortic regurgitation (AR), juvenile chronic arthritis, osteoarthritis, nephritis, tolerance induction, contact Irritability, inflammatory bowel disease (IBD), sexual dysfunction, transplantation, pain, exacerbation of HIV disease, post-inflammation depigmentation, folding screen, idiopathic droplet hypomelanosis, fever, functional bowel disease, obesity , Satiety effect, diabetes, regulation of skin exocrine function, gray hair (gray hair), gray hair, pancreatitis, fibrosis (hypertrophic scar, keloid, localized scleroderma, systemic sclerosis, Scleroderma skin graft versus host rejection, cirrhosis, idiopathic bleo Melanocortin receptors such as isine-induced pulmonary fibrosis, cyclosporine-induced nephropathy), uveitis (especially in Behcet's syndrome and sarcoidosis), vasculitis, microbial infection, celiac disease, vulvar vestibular syndrome, melanoma invasion, anorexia It is useful as a therapeutic or prophylactic agent for diseases that are responsive to the regulation of cancer.

Accordingly, one object of the present invention is to provide compounds having biological activity as described above.
Another object of the present invention is to provide a method for producing WS727713 by fermentation of a WS727713-producing strain belonging to the genus Pseudonocardia in a medium.
A further object of the present invention is to provide a pharmaceutical composition containing WS727713 as an active ingredient.
A still further object of the present invention is to provide the use of a melanocortin receptor modulator such as WS727713 for treating and preventing the diseases mentioned above.

That is, the present invention provides the following.
(1) A substantially pure WS727713 compound having the following properties:
a) Molecular formula: C ₃₀ H ₄₄ N ₈ O ₈
b) ¹ H nuclear magnetic resonance spectrum:
(500 MHz, DMSO-d6) δ
9.76 (1H, s, replaceable), 8.76 (1H, d, J = 7.5 Hz, replaceable), 8.23 (1H, d, J = 5 Hz, replaceable), 7.76 (1H, dd, J = 8.5 and 3 Hz, interchangeable), 7.25-7.17 (5H, m), 5.71 (1H, dd, J = 10.5 and 5 Hz), 5.08 (1H, dd, J = 11 and 4 Hz, interchangeable), 5.03 (1H , m), 4.99 (1H, m), 4.92 (1H, m), 4.85 (1H, m), 4.83 (1H, d, J = 3 Hz, interchangeable), 4.76 (1H, br d, J = 12 Hz, interchangeable), 4.12 (1H, dd, J = 16.5 and 8.5 Hz), 3.65 (1H, dd, J = 16.5 and 3 Hz), 3.58 (1H, m), 3.07 (1H, dd, J = 13.5 And 8.5 Hz), 2.94 (1H, br d, J = 13 Hz), 2.86 (1H, dd, J = 13.5 and 6 Hz), 2.73-2.68 (2H, m), 2.58 (1H, m), 2.24 ( 1H, br d, J = 13 Hz), 2.08 (1H, br d, J = 14 Hz), 1.88 (1H, m), 1.67 (1H, m), 1.55 (1H, m), 1.42-1.36 (2H , m), 1.21 (1H, m), 1.19 (3H, d, J = 7 Hz), 1.09 (1H, m), 0.76 (3H, d, J = 6.5 Hz), 0.75 (3H, d, J = 6.5 Hz).
c) ¹³ C nuclear magnetic resonance spectrum:
(125 MHz, DMSO-d6) δ
173.8 (s), 171.7 (s), 170.4 (s), 169.8 (s), 169.1 (s), 167.6 (s), 137.2 (s), 129.2 (d) x2, 128.0 (d) x2, 126.3 (d ), 60.2 (d), 53.2 (t), 51.1 (d), 50.8 (d), 49.6 (d), 47.4 (d), 47.0 (t), 44.7 (d), 41.2 (t), 36.4 (t ), 35.4 (t), 33.2 (t), 23.6 (d), 23.5 (t), 23.1 (q), 21.7 (q), 21.0 (t), 15.8 (q).

(2) Actinomycetes strain belonging to the genus Pseudonocardia having the deposit number FERM BP-7570.
(3) A compound having melanocortin receptor modulating activity, obtained by culturing the actinomycete strain of (2) above in a medium and recovering the compound from the culture broth.
(4) The method for producing a WS727713 compound according to (1) above, comprising culturing a WS727713-producing strain belonging to the genus Pseudonocardia in a medium and recovering the compound from the culture broth.
(5) The method of (4) above, wherein the WS727713 producing strain belonging to the genus Pseudonocardia is the actinomycete strain of (2) above.
(6) A pharmaceutical or cosmetic composition comprising the WS727713 compound of the above (1) or a salt thereof and a pharmaceutically acceptable substantially non-toxic carrier or excipient.
(7) The compound of the above (1) for use as a drug or cosmetic.
(8) A method for producing a compound having melanocortin receptor modulating activity, comprising culturing an actinomycete strain belonging to the genus Pseudonocardia that produces a compound having melanocortin receptor modulating activity in a medium and recovering the compound.
(9) A compound having melanocortin receptor modulating activity obtained by culturing an actinomycete strain belonging to the genus Pseudonocardia that produces a compound having melanocortin receptor modulating activity in a medium and recovering the compound from the culture broth.
(10) A melanocortin receptor modulator comprising the compound of the above (1) or a salt thereof.
(11) A method for modulating a melanocortin receptor comprising administering the compound of (1) or a salt thereof to a human or an animal.
(12) Use of the compound of the above (1) or a salt thereof for the manufacture of a drug or cosmetic for modulating a melanocortin receptor.

(13) An ischemia / reperfusion injury, brain and kidney containing the compound of (1) or a salt thereof and a pharmaceutically acceptable substantially non-toxic carrier or excipient Inflammatory disease, hepatitis, septic / septic shock, hypoxic shock, acute respiratory distress syndrome (ARDS), rheumatoid arthritis (RA), gouty arthritis, aortic regurgitation (AR), juvenile chronic arthritis, deformity Osteoarthritis, nephritis, tolerance induction, contact hypersensitivity, inflammatory bowel disease (IBD), sexual dysfunction, transplantation, pain, exacerbation of HIV disease, post-inflammation depigmentation, folding screen, idiopathic droplet hypomelanin Disease, fever, functional bowel disease, obesity, satiety effect, diabetes, regulation of skin exocrine function, gray hair (gray hair), gray hair, pancreatitis, fibrosis (hypertrophic scar, keloid, localized) Scleroderma, systemic sclerosis, scleroderma skin graft versus host rejection, cirrhosis, idiopathic blur Treat or prevent omycin-induced pulmonary fibrosis, cyclosporine-induced nephropathy), uveitis (especially in Behcet's syndrome and sarcoidosis), vasculitis, microbial infection, celiac disease, vulvar vestibular syndrome, melanoma invasion or anorexia Pharmaceutical or cosmetic composition for
(14) ischemia / reperfusion injury, brain and kidney inflammatory disease, hepatitis, septic / septic shock, comprising administering the compound of (1) or a salt thereof to a human or an animal, Hypoxic shock, acute respiratory distress syndrome (ARDS), rheumatoid arthritis (RA), gouty arthritis, aortic regurgitation (AR), juvenile chronic arthritis, osteoarthritis, nephritis, tolerance induction, contact hypersensitivity , Inflammatory bowel disease (IBD), sexual dysfunction, transplantation, pain, exacerbation of HIV disease, post-inflammation depigmentation, folding screen, idiopathic droplet hypomelanosis, fever, functional bowel disease, obesity, satiety Effect, diabetes, regulation of skin exocrine function, gray hair (gray hair), gray hair, pancreatitis, fibrosis (hypertrophic scar, keloid, localized scleroderma, systemic sclerosis, scleroderma Skin graft versus host rejection, cirrhosis, idiopathic bleomycin-induced pulmonary fibrosis, cyclospo Phosphorus-induced nephropathy), uveitis (especially in Behcet's syndrome and sarcoidosis), vasculitis, microbial infection, celiac disease, vulvovaginal vestibular syndrome, melanoma invasion or anorexia.
(15) Isemia / reperfusion injury, inflammatory diseases of the brain and kidneys, hepatitis, septic / septic shock, hypoxic shock, acute respiratory distress syndrome (ARDS), rheumatoid arthritis (RA), gout Osteoarthritis, aortic regurgitation (AR), juvenile chronic arthritis, osteoarthritis, nephritis, tolerance induction, contact hypersensitivity, inflammatory bowel disease (IBD), sexual dysfunction, transplantation, pain, HIV disease Exacerbation, post-inflammation depigmentation, folding screen, idiopathic droplet melanosis, fever, functional bowel disease, obesity, satiety effect, diabetes, regulation of exocrine skin function, gray hair (localized gray hair), mixed white hair Gray hair, pancreatitis, fibrosis (hypertrophic scar, keloid, localized scleroderma, systemic sclerosis, scleroderma skin graft versus host rejection, cirrhosis, idiopathic bleomycin-induced pulmonary fibrosis, cyclosporine Induced nephropathy), uveitis (especially Behcet's syndrome and sarcoid) In cis), vasculitis, microbial infections, celiac disease, vulvar vaginal vestibulitis syndrome, compounds of the above for the manufacture of a medicament for the treatment or prevention of melanoma invasion or anorexia (1) or a salt thereof.

Compounds having a strong regulatory effect on the activity of the melanocortin receptor can be identified by the following characteristics. Charts of infrared spectrum, ¹ H nuclear magnetic resonance spectrum and ¹³ C nuclear magnetic resonance spectrum are shown in FIGS.
a) Molecular formula: C ₃₀ H ₄₄ N ₈ O ₈
b) ESI-MS (+: m / z): 645 (M + H) ⁺
c) Infrared spectrum (Figure 1):
νmax (KBr): 3310, 2950, 1640, 1545, 1450, 1410, 1370, 1350,
1300, 1245, 1160, 1125, 1095, 995 cm ^-1
d) ¹ H nuclear magnetic resonance spectrum (Figure 2):
(500 MHz, DMSO-d6) δ
9.76 (1H, s, replaceable), 8.76 (1H, d, J = 7.5 Hz, replaceable), 8.23 (1H, d, J = 5 Hz, replaceable), 7.76 (1H, dd, J = 8.5 and 3 Hz, interchangeable), 7.25-7.17 (5H, m), 5.71 (1H, dd, J = 10.5 and 5 Hz), 5.08 (1H, dd, J = 11 and 4 Hz, interchangeable), 5.03 (1H , m), 4.99 (1H, m), 4.92 (1H, m), 4.85 (1H, m), 4.83 (1H, d, J = 3 Hz, interchangeable), 4.76 (1H, br d, J = 12 Hz, interchangeable), 4.12 (1H, dd, J = 16.5 and 8.5 Hz), 3.65 (1H, dd, J = 16.5 and 3 Hz), 3.58 (1H, m), 3.07 (1H, dd, J = 13.5 And 8.5 Hz), 2.94 (1H, br d, J = 13 Hz), 2.86 (1H, dd, J = 13.5 and 6 Hz), 2.73-2.68 (2H, m), 2.58 (1H, m), 2.24 ( 1H, br d, J = 13 Hz), 2.08 (1H, br d, J = 14 Hz), 1.88 (1H, m), 1.67 (1H, m), 1.55 (1H, m), 1.42-1.36 (2H , m), 1.21 (1H, m), 1.19 (3H, d, J = 7 Hz), 1.09 (1H, m), 0.76 (3H, d, J = 6.5 Hz), 0.75 (3H, d, J = 6.5 Hz).
e) ¹³ C nuclear magnetic resonance spectrum (Figure 3):
(125 MHz, DMSO-d6) δ
173.8 (s), 171.7 (s), 170.4 (s), 169.8 (s), 169.1 (s), 167.6 (s), 137.2 (s), 129.2 (d) x2, 128.0 (d) x2, 126.3 (d ), 60.2 (d), 53.2 (t), 51.1 (d), 50.8 (d), 49.6 (d), 47.4 (d), 47.0 (t), 44.7 (d), 41.2 (t), 36.4 (t ), 35.4 (t), 33.2 (t), 23.6 (d), 23.5 (t), 23.1 (q), 21.7 (q), 21.0 (t), 15.8 (q).

In particular, WS727713 has the following physicochemical characteristics:
Molecular formula:
C ₃₀ H ₄₄ N ₈ O ₈
ESI-MS (+: m / z): 645 (M + H) ⁺
HR-ESI-TOF / MS (+: m / z)
Calculated value 645.3360 (M + H) ⁺
Actual value 645.3387 (M + H) ⁺
Specific rotation:
[α] _D (23 ℃) 32 ° (c = 0.5, in DMSO)
solubility:
Soluble: methanol, acetonitrile, acetone, ethyl acetate, DMSO
Slightly soluble: H ₂ O
Color reaction Positive: iodine vapor reaction, cerium sulfate reaction,
Erich reaction, Dragendorf reaction Negative: Maurich reaction, ninhydrin reaction

Thin layer chromatography (TLC):
Stationary phase Developing solvent Rf value
Silica gel 60 F254 * CHCl ₃ : CH ₃ OH = 10: 1 0.45
RP-18W F254S HPTLC * MeCN: Water = 80: 20 0.53
* E. Merck High Performance Liquid Chromatography (HPLC):
conditions:
Mobile phase: 35% acetonitrile aqueous solution Column: Mightysil RP-18 GP (4.6 X 150mm)
Flow rate: 1.0 ml / min Detection: UV 210 nm
Retention time: 9.3 minutes Infrared spectrum (Figure 1):
νmax (KBr): 3310, 2950, 1640, 1545, 1450, 1410, 1370,
1350, 1300, 1245, 1160, 1125, 1095, 995 cm ^-1

¹ H nuclear magnetic resonance spectrum (Figure 2):
(500 MHz, DMSO-d6) δ
9.76 (1H, s, replaceable), 8.76 (1H, d, J = 7.5 Hz, replaceable), 8.23 (1H, d, J = 5 Hz, replaceable), 7.76 (1H, dd, J = 8.5 and 3 Hz, interchangeable), 7.25-7.17 (5H, m), 5.71 (1H, dd, J = 10.5 and 5 Hz), 5.08 (1H, dd, J = 11 and 4 Hz, interchangeable), 5.03 (1H , m), 4.99 (1H, m), 4.92 (1H, m), 4.85 (1H, m), 4.83 (1H, d, J = 3 Hz, interchangeable), 4.76 (1H, br d, J = 12 Hz, interchangeable), 4.12 (1H, dd, J = 16.5 and 8.5 Hz), 3.65 (1H, dd, J = 16.5 and 3 Hz), 3.58 (1H, m), 3.07 (1H, dd, J = 13.5 And 8.5 Hz), 2.94 (1H, br d, J = 13 Hz), 2.86 (1H, dd, J = 13.5 and 6 Hz), 2.73-2.68 (2H, m), 2.58 (1H, m), 2.24 ( 1H, br d, J = 13 Hz), 2.08 (1H, br d, J = 14 Hz), 1.88 (1H, m), 1.67 (1H, m), 1.55 (1H, m), 1.42-1.36 (2H , m), 1.21 (1H, m), 1.19 (3H, d, J = 7 Hz), 1.09 (1H, m), 0.76 (3H, d, J = 6.5 Hz), 0.75 (3H, d, J = 6.5 Hz).

¹³ C nuclear magnetic resonance spectrum (Figure 3):
(125 MHz, DMSO-d6) δ
173.8 (s), 171.7 (s), 170.4 (s), 169.8 (s), 169.1 (s), 167.6 (s), 137.2 (s), 129.2 (d) x2, 128.0 (d) x2, 126.3 (d ), 60.2 (d), 53.2 (t), 51.1 (d), 50.8 (d), 49.6 (d), 47.4 (d), 47.0 (t), 44.7 (d), 41.2 (t), 36.4 (t ), 35.4 (t), 33.2 (t), 23.6 (d), 23.5 (t), 23.1 (q), 21.7 (q), 21.0 (t), 15.8 (q).

Color and condition:
White crystalline melting point:
272-277 ° C (decomposition)

Characteristics of production strain No.727713
No.727713 strain was isolated from a sample of fallen leaves collected in Chiba Prefecture, Japan. Shirling and Gottlieb (Shirling, EB and D. Gottlieb: Methods for characterization of Streptomyces species. Int. J. Syst. Bacteriol. 16, 313-340, 1966) and Waksman (Waksman , SA: The actinomycetes Vol. 2: Classification, identification and description of genera and species: The Williams and Wilkins Co., Baltimore, 1961). Observation was performed after culturing at 30 ° C. for 14 days. Morphological observation was performed using light and scanning electron microscopy using humic acid-vitamin agar (Hayakawa, M. and Nonomura, H .: Humic acid-vitamin agar, a new medium for the selective isolation of soil actinomycetes J. Ferment. Technol., 65, 501-509, 1987). The temperature range for growth is 1000 ml tap water, yeast extract (Daigo Eiyo, Osaka, Japan) 2 g, soluble starch 10 g and agar 16 g agar (starch agar (pH with 1N NaOH before sterilization). Adjusted to 7.2). Carbon source utilization is Pridoham-Gottlieb medium supplemented with 0.1% yeast extract (Pridoham, TG and D. Gottlieb: The utilization of carbon compounds by some Actinomycetales as an acid for species determination: J. Bacteriol. 56: 107-114, 1948). The color names used in this study were taken from the Methuen Handbook of Color (Kornerup, A. and JH Wanscher: Methuen Handbook of Color, Methuen, London, 1978). Preparation of cells and detection of diaminopimeric acid isomers can be performed by Becker et al. (Becker, B., MP Lechevalier, RE Gordon and HA Lechevalier: Rapid differentiation between Nocardia and Streptomyces by paper chromatography of whole-cell hydrolysates. : Appl. Microbiol. 12, 421-423, 1964). Total cell sugar composition is determined by the Lechevalier-Lechevalier method (Lechevalier, MP and HA Lechevalier: Chemical composition as a criterion in the classification of aerobic actinomycetes. Int. J. Syst. Bacteriol., 20, 435-443, 1970) did. 16S rDNA sequence was determined by the method of Reichert et al. (Katrin Reichert, Andre Lipski, Silke Pradella, Erko Stackebrandt and Karlheinz Altendorf: Pseudonocardia asaccharolytica sp. Nov. And Pseudonocardia sulfidoxydans sp. the genus Pseudonocardia: Int. J. Syst. Bacteriol. 48, 441-449, 1998). The strain type 16S rDNA sequence was obtained from the DDBJ database (http://www.ddbj.nig.ac.jp). The phylogenetic tree is the CLUSTAL program (Thompson, JD, Gibson, TJ, Plewniak, F., Jeanmougin, F. and Higgins, DG: The CLUSTAL X windows interface: flexible stradesies for multiple sequence alignment aided by quality analysis tools.Nucleic Acids Res 24: 4876-4882, 1997).

The bacteriological characteristics were as follows:
Culture characteristics and physiological characteristics on various agar media are shown in Tables 1 and 2, respectively. Strain growth was observed on yeast extract-malt extract agar, oatmeal agar and peptone-yeast extract-iron agar, but not on inorganic salt-starch agar, glycerol-asparagine agar and tyrosine agar. The aerial mycelium was observed only on humic acid-vitamin agar, and the color of the aerial mycelium was white. The color of the back of the growth was light orange. Melanoid pigments were not produced in tyrosine-yeast extract broth and peptone-yeast extract-iron agar. No soluble pigment was produced. The intracellular pigment was not pH sensitive.

As a morphological feature, the basic hyphae grew well and branched irregularly. The formation of swollen mycelium pieces was observed. This strain produced aerial mycelium with straight spore chains and more than 10 spores per chain. The spore shape was a spindle (1.6-2.3 × 0.5-0.6 μm). Their surfaces were smooth. Spherical granules, sporangia and motile spores or fragments were not observed.
The No. 727713 strain was able to grow in the temperature range of 11 to 32 ° C., and the optimal growth condition was 28 ° C.
Meso-diaminopimelic acid, lactose and arabinose were detected in whole cell hydrolysates of this strain and the cell wall type was type IV.
The partial sequence of No. 727713 strain is shown as Sequence ID NO: 1 in the Sequence Listing. The 16S rDNA homology values between No.727713 strain and members of the genus Pseudonocardia are 91.8-97.4%, making them a single cluster in the phylogenetic tree (data not shown).

  Based on the morphological and chemical characteristics and the phylogenetic analysis described above, the No.727713 strain is recognized as belonging to the genus Pseudonocardia (Lechevalier, MP and HA Lechevalier: Chemical composition as a criterion in the clasification of aerobic actinomycetes. Int. J. Syst. Bacteriol., 20, 435-443, 1970; Pridham, TG, et al .: Appl. Microbiol. 6: 54, 1958). Therefore, this strain was defined as Pseudonocardia sp. No.727713. This strain was deposited as FERM BP-7570 at AIST Tsukuba Chuo 6 305-8566, AIST Tsukuba Chuo 6-1, 1-chome Higashi 1-chome, Tsukuba, Ibaraki, Japan, National Institute of Advanced Industrial Science and Technology (Deposited date: 2001) April 25).

  These features were observed after 14 days incubation at 30 ° C. Color descriptions were based on the Methhuen Handbook of Color (Kornerup, A. and J. H. Wanscher, 3rd ed., Pp. 252, Methuen, London, 1978).

  It should be understood that the production of WS727713 is not limited to the use of the specific organisms described herein, which are given for illustrative purposes only. The present invention relates to artificial mutants that can be produced from organisms described by conventional means such as recombinant DNA technology, X-ray irradiation, ultraviolet irradiation, N-methyl-N′-nitro-N-nitrosoguanidine, 2-aminopurine, etc. As well as the use of any mutants capable of producing WS727713, including natural mutants.

Production of WS727713 Among the compounds having the characteristics described above, WS727713 production strain belonging to Pseudonocardia grows in medium containing assimilable carbon and nitrogen sources under aerobic conditions (eg shaking culture, submerged culture, etc.) If you do, WS727713 will be produced.
A preferred carbon source in the medium is a carbohydrate such as glucose, sucrose, starch, fructose or glycerin.
Preferred nitrogen sources are ammonium salts (eg, ammonium nitrate, ammonium sulfate, ammonium phosphate, etc.), inorganic and organic nitrogen compounds such as urea or amino acids, and peanut powder, yeast extract, beef extract, peptone, polypeptone, gluten meal, cottonseed flour , Soybean powder, soybean meal, corn steep liquor, dry yeast, wheat germ and the like.

  Carbon and nitrogen sources are advantageous when used in combination, but impure materials containing trace amounts of growth factors and large amounts of inorganic nutrients are also suitable for use, so it is not necessary to use them in their pure form .

When desired, inorganic salts such as sodium carbonate or calcium carbonate, sodium phosphate or potassium phosphate, sodium chloride or potassium chloride, sodium iodide or potassium iodide, magnesium salt, copper salt, zinc salt, iron salt or cobalt salt It may be added to the medium.
If necessary, an antifoaming agent such as liquid paraffin, fatty oil, vegetable oil, mineral oil, silicone, etc. may be added, especially when the culture medium is seriously foamed.

Agitation and aeration of the culture mixture can be accomplished by various methods, such as agitation with a propeller or similar mechanical agitation device, agitation by rotation or shaking of a fermenter.
Fermentation may vary according to fermentation conditions and scale, but is usually carried out at a temperature between about 10 ° C. and 40 ° C., preferably 20 ° C. to 30 ° C., over a period of about 50 hours to 150 hours.
The resulting culture broth can then be used for biological recovery such as solvent extraction with a suitable solvent or mixture of several solvents, chromatography or recrystallization from a suitable solvent or mixture thereof for the recovery of WS727713. It is subjected to various processes conventionally used for material recovery and purification.

WS727713 may be in the form of a solvate that is within the scope of the present invention. Preferred solvates include hydrates and ethanolates.
As examples showing the biological characteristics of the above compounds, some biological data are given below.

Test 1. Binding assay Binding buffer is 25 mM HEPES-KOH (pH 7.0), 1.5 mM CaCl ₂ , 1 mM MgSO ₄ , 100 mM NaCl, 1 mM 1,10-phenanthroline, 0.2% BSA and complete TM protease inhibitor (Boehringer-Mannheim) 1 tablet / 100 mL. The binding assay was modified from the method of Receptor Biology, Inc. Membrane suspension (25 ml), [ ¹²⁵ I] -NDP-MSH (25 ml, NEN, final 3.08 × 10 ⁻¹¹ M), buffer (50 ml) and inhibitor were incubated at 37 ° C. for 60 minutes. The mixture was then filtered through GF / C (presoaked with 0.5% polyethyleneimine) and washed twice with ice-cold PBS (−). The radioactivity trapped on the GF / C filter was measured using a Top Counter (Packard) after drying.

  B16 cells were grown in RPMI 1640 supplemented with 10% FBS and antibiotics. Sub-confluent cells were washed twice with ice-cold PBS (-) and scraped. The cells were then sonicated and centrifuged at 2,000 rpm for 5 minutes at 4 ° C. The supernatant thus obtained was centrifuged at 30,000 rpm for 60 minutes at 4 ° C. The membrane fraction was suspended in ice-cold PBS (−) and stored at −80 ° C.

  The final concentration of each receptor protein was as follows: MC1-R (B16), 0.139 mg / ml; MC3-R (recombinant *), 0.0693 mg / ml; MC4-R (recombinant * ), 0.104 mg / ml; and MC5-R (recombinant *), 0.070 mg / ml (* from Receptor Biology, Inc.)

As shown in Table 3, WS727713 inhibited [ ¹²⁵ I] -NDP-MSH binding to the melanocortin receptor. This result indicates that WS727713 acts as an agonist or antagonist of the melanocortin receptor.

Test 2. Dose-dependent reduction of LPS-induced TNF-α production in mice by WS727713 Tumor necrosis factor (TNF-α) underlies pathological processes and functional impairment in acute and chronic inflammatory diseases and injuries. The neuroimmunomodulatory peptide α-MSH regulates the action and production of inflammatory cytokines, including TNF-α. To test for WS727713, in two replicates, either lipopolysaccharide in saline (LPS; Sigma, L-3129, E.coli 0127: B8, 100 mg / head) or saline alone Were injected intraperitoneally into male mice (ICR). WS727713 was injected intraperitoneally 30 minutes before LPS injection. Mice were sacrificed 60 minutes after LPS injection. Serum TNF-α levels were measured using enzyme-linked immunosorbent assay (TNF-α ELISA kit; Amersham). The results are shown in Table 4.

  As shown in Table 4, LPS-induced TNF-α levels were reduced by administration of WS727713 in a dose-dependent manner. The results indicate that WS727713 has a function as a melanocortin receptor agonist.

  The substantially pure compound WS727713 is a modulator of the melanocortin receptors MC1-R, MC3-R, MC4-R, and / or MC5-R, and in particular has agonist activity of MC1-R. A pharmaceutical composition containing the substantially pure compound WS727713 is useful as a therapeutic or prophylactic agent for diseases caused by inflammation or immune conditions. Furthermore, it is useful for treating inflammation, in particular inflammation caused by activation of NF-kB and / or release of inflammatory cytokines. In addition, pharmaceutical formulations containing substantially pure compound WS727713 are useful for the treatment and prevention of the following diseases.

  Inflammatory bowel disease, irritable bowel syndrome, celiac disease, gallbladder disease, Crohn's disease, ulcerative colitis, rheumatoid arthritis, gouty arthritis, osteoarthritis, osteoporosis, traumatic arthritis, rubella arthritis, muscle degeneration, Pancreatitis (acute or chronic), psoriasis, glomerulonephritis, serum sickness, lupus (systemic lupus erythematosus), urticaria, scleraclerma, scleroderma, chronic thyroiditis, Graves' disease, dermatitis (contact or atopy), external Vulvar vestibular syndrome, dermatomyositis, alopecia, atopic eczema, ichthyosis, fever, sepsis, migraine, cluster headache, Alzheimer's disease, Parkinson's disease, Creutzfeldt-Jakob disease, multiple sclerosis, tuberculosis, Dementia and transplant or graft versus host rejection (e.g. kidney, liver, heart, lung, pancreas, bone marrow, cornea, small intestine, skin allograft, skin allograft and xenograft Diseases associated with chronic and acute inflammation, such as hemisphere), and immune modulation.

  Respiratory allergies and diseases such as asthma, acute respiratory distress syndrome, hay fever, allergic rhinitis, and chronic obstructive pulmonary disease; central nervous system inflammation such as HIV encephalitis, cerebral malaria, meningitis, and telangiectasia Sexual disorders; and pain such as postoperative pain, neuromuscular pain, headache, cancer pain, toothache and joint pain.

  Herpes simplex type 1 (HSV-1), herpes simplex type 2 (HSV-2), cytomegalovirus, Epstein Barr, human immunodeficiency virus (HIV), Addison's disease (adrenal autoimmune disease), idiopathic adrenal insufficiency , Autoimmune multiglandular disease (also known as autoimmune multiglandular syndrome), chronic active hepatitis or acute hepatitis infection (such as hepatitis A, hepatitis B and hepatitis C), autoimmune gastritis, self Viruses and autoimmune diseases such as immune hemolytic anemia and autoimmune neutropenia; and fungal infections such as mycosis fungoides.

  Atherosclerosis, transplant atherosclerosis, peripheral vascular disease, inflammatory vascular disease, intermittent claudication, restenosis, cerebral vascular stroke, transient ischemic stroke, myocardial ischemia and myocardial infarction. Also circulatory diseases such as hypertension, hyperlipidemia, coronary artery disease, unstable angina, thrombosis, thrombin-induced platelet aggregation, and / or pathology resulting from the formation of thrombosis and / or atherosclerotic plaque.

  Stroke and other ischemic brain diseases and / or neurodegeneration associated therewith, and neurodegeneration of traumatic brain injury or its consequences.

  Sunburn, acne, vitiligo, alopecia arreata, photosensitivity disorder, pigment deficiency, gray hair (gray hair), gray hair, dry skin and porphyria.

  Neurodegenerative disorders such as depression, anxiety, obsessive compulsion (obsessive compulsive disorder), neurosis, psychosis, insomnia / sleep disorder, sleep apnea, and drug or substance abuse.

  Male sexual dysfunction, including sexual inability, decreased libido, and erectile dysfunction (such as inability to achieve or maintain erection, inability to ejaculate, premature ejaculation, inability to achieve orgasm). Women with sexual arousal disorder or sexual desire, sexual acceptability, orgasmus, and / or disorders related to impaired sexual function, and sexual pain, premature birth, dysmenorrhea, menorrhagia and endometriosis Sexual dysfunction.

  Body weight disorders such as obesity and anorexia (eg, due to changes in appetite, metabolic rate, fat intake or carbohydrate craving); and diabetes (due to increased glucose tolerance and / or decreased insulin resistance).

  Neoplastic disorders such as lung, prostate, colon, breast, ovarian and bone cancer, skin cancer (eg, melanoma invasion), or solid tumor formation or growth.

  Fibrosis such as hypertrophic scars, keloids, localized scleroderma, systemic sclerosis, scleroderma skin graft versus host rejection, cirrhosis, idiopathic bleomycin-induced pulmonary fibrosis, and cyclosporine-induced nephropathy.

  Veterinary diseases such as livestock virus infections such as feline immunodeficiency virus, bovine immunodeficiency virus and canine immunodeficiency virus.

  Uveitis (especially in Behcet's syndrome and sarcoidosis), vasculitis and microbial infection.

  The pharmaceutical and cosmetic compositions of the present invention are compounds as active ingredients in mixtures with organic or inorganic carriers or excipients suitable for topical, enteral or parenteral administration, eg in solid, semi-solid or liquid form. Used in the form of a pharmaceutical preparation containing a melanocortin receptor modulator such as WS727713. Active ingredients are suitable for eg tablets, pellets, capsules, suppositories, solutions, emulsions, suspensions, injections, ointments, liniments, eye drops, lotions, eye drops, lotions, gels, creams and uses For other forms, it can be mixed with pharmaceutically acceptable carriers that are usually non-toxic.

  Carriers that can be used are water, glucose, lactose, acacia gum, gelatin, mannitol, starch paste, magnesium trisilicate, talc, corn starch, keratin, colloidal silica, potato starch, urea in solid, semi-solid or liquid form And other carriers suitable for use in preparing the formulations. In addition, adjuvants, stabilizers, thickeners, solubilizers and coloring agents and fragrances may be used.

  In order to apply the composition to humans, it is preferably applied by intravenous, intramuscular, topical or oral administration. On the other hand, the dosage of a therapeutically effective amount of a melanocortin receptor modulator such as compound WS727713 will vary and depend on the age and condition of the individual patient being treated. When individual patients are treated, when administered intravenously, melanocortin receptor modulators such as compound WS727713 daily dose 0.001-100 mg / kg body weight (human), and when administered intramuscularly, melanocortin receptors such as compound WS727713 Daily dosage of 0.001-100 mg / kg body weight of human body modulator (human), and oral administration of 0.01-320 mg / kg body weight of human melanocortin receptor modulator such as compound WS727713 for normal treatment To give.

Examples The following examples are given for the purpose of illustrating the invention in more detail.

Example 1
(1) Fermentation of Pseudonocardia sp. No. 727713 for production of WS727713:
Platinum eard slant cultures are sterilized seed culture medium containing 2% soluble starch, 0.8% yeast extract and 0.5% agar in a wide-mouthed Erlenmeyer flask (225 ml) adjusted to pH 7.0 before sterilization 60 Planted in ml. The flasks were incubated for 7 days at 30 ° C. on a rotary shaker (220 rpm, 5.1 cm-throw) and then each planted in 80 ml of the same sterilized medium in 5 wide-mouthed Erlenmeyer flasks (225 ml) (2% ). The flask was incubated for 4 days at 30 ° C. on a rotary shaker (220 rpm, 5.1 cm-throw).

The resulting seed culture was planted (2%) in 20 liters of sterile production medium in a 30 liter jar fermenter. Production medium is 1% glucose, 1.5% soluble starch, 0.5% yeast extract, 1% β-cyclodextrin, 1% CaCO ₃ , 0.05% Adecanol LG-109 (Asahi Denka Kogyo Co., Ltd., Japan) and 0.05% Silicone KM -70 (Shin-Etsu Chemical Co., Japan) (adjusted to pH 7.0 before sterilization). Fermentation was carried out at 28 ° C. for 5 days with aeration of 20 L / min and stirring at 200 rpm.
The production of WS727713 in the fermentation broth was measured by the HPLC analysis shown below.

(Analytical HPLC conditions)
Column Mightysil RP-18 GP 150-4.6, 5 mm
(5 μm, 4.6 mm ID × 150 mm L, Kanto Chemical Co., Japan)
Eluent CH ₃ CN: H ₂ O = 35: 65
Flow rate 1 ml / min Temperature Room temperature Detection UV 210 nm
Retention time 9.3 minutes

(2) Isolation of WS727713:
After completion of the culture, the culture broth (16 L) was extracted by stirring with an equal volume of acetone for several minutes at room temperature. The extract was filtered using diatomaceous earth. The filtrate was concentrated in vacuo to give an aqueous solution (15 L). The solution was applied to a Diaion HP20 (Mitsubishi Chemical Corporation) column (800 ml), and the column was washed with 20% acetonitrile (4 L). The column was eluted with 40% acetonitrile (2.4 L) and 60% acetonitrile (3.6 L). The eluate (6.0 L) was diluted to 12 L with water and applied to a Daisogel SP-120-ODS-B column (1 L) (15/30 μm, Daiso Corporation, Japan) filled with water. The column was eluted with 32.5% acetonitrile. Elution was measured by analytical HPLC as indicated above. Fractions containing WS727713 were collected and concentrated in vacuo to give a powder. This powder was dissolved in a small amount of methanol and crystallized from water. The crystals were filtered and dried to give 1.5 g of WS727713 as white needles.

  The novel compound of the present invention has melanocortin receptor modulating activity, is useful as an active ingredient of anti-inflammatory agents, and is useful as a therapeutic or prophylactic agent for diseases that respond to melanocortin receptor modulation.

  This application is based on patent application No. 2004901919 filed in Australia, the contents of which are incorporated in full herein.

FIG. 1 shows an infrared spectrum chart. FIG. 2 shows a chart of ¹ H nuclear magnetic resonance spectrum. FIG. 3 shows a chart of ¹³ C nuclear magnetic resonance spectrum.

Claims (10)

A substantially pure WS727713 compound having the following properties:
a) Molecular formula: C ₃₀ H ₄₄ N ₈ O ₈
b) ¹ H nuclear magnetic resonance spectrum:
(500 MHz, DMSO-d6) δ
9.76 (1H, s, replaceable), 8.76 (1H, d, J = 7.5 Hz, replaceable), 8.23 (1H, d, J = 5 Hz, replaceable), 7.76 (1H, dd, J = 8.5 and 3 Hz, interchangeable), 7.25-7.17 (5H, m), 5.71 (1H, dd, J = 10.5 and 5 Hz), 5.08 (1H, dd, J = 11 and 4 Hz, interchangeable), 5.03 (1H , m), 4.99 (1H, m), 4.92 (1H, m), 4.85 (1H, m), 4.83 (1H, d, J = 3 Hz, interchangeable), 4.76 (1H, br d, J = 12 Hz, interchangeable), 4.12 (1H, dd, J = 16.5 and 8.5 Hz), 3.65 (1H, dd, J = 16.5 and 3 Hz), 3.58 (1H, m), 3.07 (1H, dd, J = 13.5 And 8.5 Hz), 2.94 (1H, br d, J = 13 Hz), 2.86 (1H, dd, J = 13.5 and 6 Hz), 2.73-2.68 (2H, m), 2.58 (1H, m), 2.24 ( 1H, br d, J = 13 Hz), 2.08 (1H, br d, J = 14 Hz), 1.88 (1H, m), 1.67 (1H, m), 1.55 (1H, m), 1.42-1.36 (2H , m), 1.21 (1H, m), 1.19 (3H, d, J = 7 Hz), 1.09 (1H, m), 0.76 (3H, d, J = 6.5 Hz), 0.75 (3H, d, J = 6.5 Hz).
c) ¹³ C nuclear magnetic resonance spectrum:
(125 MHz, DMSO-d6) δ
173.8 (s), 171.7 (s), 170.4 (s), 169.8 (s), 169.1 (s), 167.6 (s), 137.2 (s), 129.2 (d) x2, 128.0 (d) x2, 126.3 (d ), 60.2 (d), 53.2 (t), 51.1 (d), 50.8 (d), 49.6 (d), 47.4 (d), 47.0 (t), 44.7 (d), 41.2 (t), 36.4 (t ), 35.4 (t), 33.2 (t), 23.6 (d), 23.5 (t), 23.1 (q), 21.7 (q), 21.0 (t), 15.8 (q).   Actinomycetes strain belonging to the genus Pseudonocardia having the deposit number FERM BP-7570.   The compound of claim 1 obtained by culturing the actinomycete of claim 2 in a medium and recovering the compound from the culture broth.   The method for producing a WS727713 compound according to claim 1, comprising culturing a WS727713-producing strain belonging to the genus Pseudonocardia in a medium and recovering the compound from the culture broth.   The method of claim 4, wherein the WS727713 producing strain belonging to the genus Pseudonocardia is the actinomycete of claim 2.   A pharmaceutical composition comprising the WS727713 compound of claim 1 or a salt thereof and a pharmaceutically acceptable substantially non-toxic carrier or excipient.   A compound of claim 1 or a salt thereof for use as a medicament or cosmetic. In a medium, wherein producing the compound of claim 1 by culturing the actinomycetes strain belonging to the genus Pseudonocardia, compound of claim 1 which further obtained recovering the compound from the culture broth.   A melanocortin receptor modulator comprising the compound of claim 1 or a salt thereof.   Use of a compound of claim 1 or a salt thereof for the manufacture of a medicament or cosmetic for modulating a melanocortin receptor.

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